The invention relates to methods of reducing cancer growth in biological systems. More specifically, the invention relates to the inhibition of solid tumor invasiveness and metastasis in mammals.
Cancer, in all of its myriad manifestations, remains a devastating scourge upon mankind. While progress in preventing and treating cancer has been made, including particular success against Hodgkin""s lymphoma and certain other forms, many types of cancer remain substantially impervious to prevailing treatment protocols. Typically, cancer is treated by chemotherapy, in which highly toxic chemicals are given to the patient, or by radiotherapy, in which toxic doses of radiation are directed at the patient. While commonly effective to kill huge numbers of cancer cells, these xe2x80x9ccytotoxicxe2x80x9d treatments also kill extraordinary numbers of healthy cells, causing the patient to experience acute debilitating symptoms including nausea, diarrhea, hypersensitivity to light, hair loss, etc. The side effects of these cytotoxic compounds limits the frequency and dosage at which they can be administered. Such disabling side effects can be mitigated to some degree by using compounds that selectively target cycling cells, i.e., interfering with DNA replication or other growth processes in cells that are actively reproducing. Since cancer cells are characterized by their extraordinary ability to proliferate, such protocols preferentially kill a larger proportion of cancer cells in comparison to healthy cells, but cytotoxicity and ancillary sickness remains a problem.
Other more recent developments include efforts to develop monoclonal antibodies specific for oncogenes or HLA specificities, to identify cancer cells with great precision. However, these procedures are very expensive and extremely procedurally elaborate, yet still fail to produce the desired efficacy. Indeed, such procedures have been reported to be effective in only a small subpopulation of treated patients.
The area of cancer research concerned with the mechanisms of tumor cell invasion has benefited greatly from the conceptual framework proposed by Liotta and colleagues (see, e.g., Yamamoto et al. (1996); Emmert-Buck et al. (1994). This model describes the invasive process as a logical progression of events involving three discernible stages: attachment of tumor cells to an extracellular matrix (ECM), proteolytic digestion of the matrix, and movement of cells through the proteolytically degraded barrier. A key factor in this process is the regulation of the matrix metalloproteinases (MMPs; including gelatinases A and B; MMP-2 and MMP-9, respectively, and MMP-3 (Lokeshwar et al. 1993a)), that play a major role in the degradation of the ECM during invasion.
Tetracycline and a number of its chemical relatives form a particularly successful class of antibiotics. Certain of the tetracycline compounds, including tetracycline itself, as well as sporocycline, etc., are broad spectrum antibiotics, having utility against a wide variety of bacteria. The parent compound, tetracycline, has the following general structure: 
The numbering system for the multiple ring nucleus is as follows: 
Tetracycline, as well as the 5-OH (terramycin) and 7-Cl (aureomycin) derivatives, exist in nature, and are all well known antibiotics. Semisynthetic derivatives such as 7-dimethylamino-tetracycline (minocycline) and 6xcex1-deoxy-5-hydroxy-tetracycline (doxycycline) are also known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained to do so. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher (1978). According to Mitscher, modification at positions 5-9 of the tetracycline ring system can be made without causing the complete loss of antibiotic properties.
However, changes to the basic structure of the ring system, or replacement of substituents at positions 1-4 or 10-12, generally lead to synthetic tetracyclines with substantially less, or essentially no, antibacterial activity. For example, 4-de(dimethylamino)tetracycline is commonly considered to be a non-antibacterial tetracycline.
More recently, it has been established that tetracyclines, which are rapidly absorbed and have a prolonged plasma half-life, exert biological effects independent of their antimicrobial activity (Golub et al. 1991, Golub et al. 1992, Uitto et al. 1994). Such effects include inhibition of matrix metalloproteinases (abbreviated xe2x80x9cMMPsxe2x80x9d), including collagenases (MMP-1; MMP-8; MMP-13) and gelatinases (MMP-2; MMP-9), as well as prevention of pathologic tissue destruction (Golub et al. 1991). Recent studies have suggested that, in some systems, certain tetracyclines and inhibitors of metalloproteinases can inhibit tumor progression (DeClerck et al. 1994) or angiogenesis (WIPO publication WO 92/12717; Maragoudakis et al. 1994). Zucker et al. (1985) showed that minocycline can inhibit melanoma cell activity in vitro. Some tetracyclines may exhibit cytotstatic effects against some tumors (Kroon et al. 1984; van den Bogert et al. 1986).
However, the use of tetracycline antibiotics, while generally effective for treating infection, can lead to undesirable side effects. For example, the long term administration of antibiotic tetracyclines can reduce or eliminate healthy microbial flora, such as intestinal flora, and can lead to the production of antibiotic resistant organisms or the overgrowth of yeast and fungi. Accordingly, chemically-modified tetracyclines, in which the antimicrobial activity is attenuated or deleted, can be preferred for use in applications in which anti-collagenolytic activity is indicated.
In view of the above considerations, it is clear that there is a need to supplement existing methods of inhibiting cancer cell invasiveness and metastasis. Current approaches rely on highly cytotoxic compounds that cause ancillary debilitating sickness in patients, or use methodology that is expensive, procedurally difficult, and unpredictable.
Accordingly, it is one of the purposes of this invention to overcome the above limitations in cancer treatment, by providing a compound and method for inhibiting the growth processes characteristic of cancer cells, including inhibiting invasiveness and metastasis, as well as inducing regression of primary tumors. In particular, it is desirable to identify new anticancer compounds and methods that inhibit cancer growth specifically and with relatively high activity, i.e., being active at doses that are substantially free of harmful side effects.
It has now been discovered that these and other objectives can be achieved by the present invention, which provides a method for inhibiting the growth or development of cancer in a mammal by providing a chemically modified tetracycline to the mammal in an amount that is effective to achieve the specified result.
In one embodiment, the invention is a method of inhibiting cancer growth in a mammal, comprising administering to the mammal a cancer-inhibitory amount of a tetracycline compound selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1),
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
tetracycline pyrazole (CMT-5),
6xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-12xcex1-deoxyanhydrotetracycline (CMT-9), and
4-de(dimethylamino)minocycline (CMT-10).
A highly preferred tetracycline compound is
6-demethyl-6-deoxy-4-dedimethylaminotetracycline (CMT-3)
The method is useful for inhibiting growth of cancers such as carcinomas, e.g., carcinomas of the lung, prostate, breast, ovary, testes, or colon, as well as melanomas.
The method can comprise inhibiting cellular proliferation of the cancer, inhibiting invasiveness of the cancer, and/or inhibiting metastasis of the cancer.
The tetracycline compound can be administered in an amount sufficient to specifically inhibit expression of a matrix metalloproteinase by cells of the cancer or its activity in the extracellular matrix.
In a preferred aspect, the method is useful to inhibit a matrix metalloproteinase which is a gelatinase, such as gelatinase A or gelatinase B.
The method can further comprise treating the mammal with an adjunct antineoplastic modality. The adjunct antineoplastic modality can comprise chemotherapy, surgery, and/or radiotherapy.
In another embodiment, the invention is a method of inhibiting cancer growth in a mammal, comprising administering to the mammal a cancer-inhibitory amount of a tetracycline compound selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1),
tetracyclinonitrile (CMT-2),
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-12xcex1-deoxyanhydrotetracycline (CMT-9), and
4-de(dimethylamino)minocycline (CMT-10).
In another embodiment, the invention is a method of inhibiting proliferation of cancer cells, comprising contacting the cancer cells with a proliferation-inhibitory amount of a tetracycline compound selected from the group consisting of:
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)tetracycline (CMT-1),
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), and
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7).
Preferably, the tetracycline compound is 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) or 6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8).
In another embodiment, the invention is a method of inhibiting the invasive potential of cancer cells, comprising contacting the cancer cells with an invasion-inhibitory amount of a tetracycline compound selected from the group consisting of:
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)tetracycline (CMT-1),
4-de(dimethylamino)-12-deoxytetracycline (CMT-7),
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8), and tetracyclinonitrile (CMT-2).
Preferably, the tetracycline compound is 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).
In still another embodiment, the invention is a method of inhibiting the metastatic potential of cancer cells, comprising contacting the cancer cells with a metastasis-inhibitory amount of a tetracycline compound selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1) and
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).
In yet another embodiment, the invention is a method of treating a cancer condition characterized by excessive gelatinolytic activity, comprising administering to a mammal an amount of a tetracycline compound effective to inhibit excessive gelatinolytic activity.
In this embodiment, the cancer may be characterized by excessive activity of gelatinase A, and the tetracycline compound is selected from the group consisting of:
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7),
6-demethyl4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)tetracycline (CMT-1),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and
tetracyclinonitrile (CMT-2).
More preferably, the tetracycline compound is
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7), or
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).
Alternatively, in this embodiment, the cancer condition may be characterized by excessive activity of gelatinase B, and the tetracycline compound is selected from the group consisting of:
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
4-de(dimethylamino)tetracycline (CMT-1), and
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6).
More preferably, in this case the tetracycline compound is
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) or
4-de(dimethylamino)-7-chlorotetracycline (CMT-4).
In yet another embodiment, the invention is a method of inhibiting tumor incidence in a mammal, comprising
(a) detecting in a biological sample from the mammal a gene product or metabolite associated with predisposition to a cancer prior to observing any specific cancerous lesion; and
(b) administering to the mammal a tumor incidence-inhibiting amount of a tetracycline compound selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1),
tetracyclinonitrile (CMT-2),
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4), and
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-12xcex1-deoxyanhydrotetracycline (CMT-9), and
4-de(dimethylamino)minocycline (CMT-10).
In still another embodiment, the invention is a method of inhibiting gelatinolytic activity associated with a cancerous tumor in a mammal, comprising administering to the mammal an amount of a tetracycline compound effective to inhibit gelatinolytic activity.
The gelatinolytic activity may derive from the cancerous tumor, or it may derived from normal tissue, or both. If normal tissue is involved, the normal tissue may be epithelial tissue or stromal tissue.
In yet another embodiment, the invention is a method of inhibiting cancer growth in a mammal, comprising topically administering to the mammal a cancer-inhibitory amount of a tetracycline compound selected from the group consisting of:
tetracyclinonitrile (CMT-2) and
4-hydroxy-4-dedimethylaminotetracycline (CMT-6).
In another embodiment, the invention is a method of killing cancer cells, comprising contacting cancer cells with a cytotoxic amount of a tetracycline compound selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1),
tetracyclinonitrile (CMT-2),
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-12xcex1-deoxyanhydrotetracycline (CMT-9), and
4-de(dimethylamino)minocycline (CMT-10).
In this embodiment, the cancer cells can be cells of a sarcoma, including Kaposi""s sarcoma, or of a carcinoma, such as an adenocarcinoma. For example, the method can be used to kill cells of a carcinoma of the prostate, breast, ovary, testis, lung, colon, or breast. Preferably, the cancer cells are cells of a carcinoma of the prostate, and the tetracycline compound is 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3).
In yet another embodiment, the invention is a method of inhibiting the growth of a cancer in a mammal, comprising administering to a mammal having a cancer an amount of a tetracycline compound sufficient to induce differential cytotoxicity in cells of the cancer, wherein the tetracycline compound is selected from the group consisting of:
4-de(dimethylamino)tetracycline (CMT-1),
tetracyclinonitrile (CMT-2),
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),
4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
4-de(dimethylamino)-12xcex1-deoxytetracycline (CMT-7),
6-xcex1-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),
4-de(dimethylamino)-12xcex1-deoxyanhydrotetracycline (CMT-9), and
4-de(dimethylamino)minocycline (CMT-10).
By means of the invention, a method of killing cancer cells or inhibiting cancer growth or metastasis is provided that further avoids or mitigates side effects commonly associated with antineoplastic chemotherapeutic regimens. These and other advantages of the present invention will be appreciated from the detailed description and examples set forth hereinbelow. The detailed description and examples enhance the understanding of the invention, but are not intended to limit the scope of the invention.